The present invention relates to an image reading apparatus and, particularly to such an apparatus which can be used in a fluorescence detecting system and can accurately read a fluorescence image.
A fluorescence detecting system using a fluorescent substance as a labeling substance is known. According to this system, it is possible to study a genetic sequence, the expression level of a gene and the metabolism, absorption, excretion path and state of a substance introduced into a test animal and to effect the separation or identification of protein or the estimation of the molecular weight or properties of protein or the like by the steps of labeling a specimen using the fluorescent substance, irradiating the specimen with radiation, exciting the fluorescent substance contained in the specimen and detecting the released fluorescent light. For example, this system can perform a process including the steps of electrophoresing a plurality of DNA fragments on the gel support after a fluorescent dye was added to a solution containing a plurality of DNA fragments to be electrophoresed or distributing a plurality of DNA fragments on a gel support containing fluorescent dye or dipping a gel support on which a plurality of DNA fragments have been electrophoresed in a solution containing fluorescent dye, thereby labeling the electrophoresed DNA fragments, exciting the fluorescent dye by a stimulating ray to cause it to release a fluorescent light, detecting the released fluorescent light to produce an image and detecting the distribution of the DNA fragments on the gel support. This system also performs a process including the steps of electrophoresing a plurality of DNA fragments on a gel support, denaturing the DNA fragments, transferring at least a part of the denatured DNA fragments onto a transfer support such as a nitrocellulose support by the Southern-blotting method, hybridizing a probe prepared by labeling target DNA and DNA or RNA complementary thereto with the denatured DNA fragments, thereby selectively labeling only DNA fragments complementary to the probe DNA or probe RNA, exciting the fluorescent dye by a stimulating ray to cause it to release a fluorescent light, detecting the fluorescent light to produce an image and detecting the distribution of the target DNA fragments on the transfer support. This system can further perform a process including the steps of preparing a DNA probe complementary to DNA containing a target gene labeled by a labeling substance, hybridizing it with DNA on a transfer support, combining an enzyme with complementary DNA labeled by a labeling substance, causing the enzyme to contact a fluorescent substance, transforming the fluorescent substrate to a fluorescent substance having a property to release fluorescent light, exciting the thus produced fluorescent substance by a stimulating ray to release fluorescent light, detecting the fluorescent light to produce an image and detecting the distribution of the target DNA fragment on the transfer support. This fluorescent detecting system is advantageous in that a genetic sequence or the like can be easily detected without using a radioactive substance.
Since the gel support often used in such a fluorescence detecting system contains much water and is significantly soft, the surfaces of the gel support are not usually flat and distribution of water attached to the surfaces is not uniform. Due to the uneven (not flat) surfaces of the gel support and the uneven distribution of water on the surfaces of the gel support, it is difficult to accurately read the fluorescence image from the gel support when the detection of the fluorescent light released from the fluorescent substrate is performed by downwardly irradiating the top surface of the gel support with the stimulating light.
Therefore, there has been proposed an image reading apparatus in which an image carrier, such as a gel support or a transfer support, is placed on a flat base made of transparent material such as glass or the like to flatten its bottom surface and to scan the flattened bottom surface thereof through the flat base by a stimulating ray such as a laser beam to excite the fluorescent substance contained in the image carrier and to release fluorescent light. The fluorescent light thus released is photoelectrically detected by detecting means provided below the base.
The above-described fluorescence detecting system may perform a process including the steps of labeling specimens with a fluorescent substance, putting the labeled specimens into wells of a micro-plate disclosed in, for example, Japanese Laid-Open Publication No.6-186339, projecting a stimulating ray onto the specimen and detecting fluorescent light from the fluorescent substance. In this case, since the volume of the specimens tends to be different between different wells the vertical position from which the fluorescent light is released may differ among the wells. As a result, it may be meaningless to quantitatively compare the intensity of the fluorescent light from one well with that from the other wells and to effect quantitative analysis of the fluorescent images from the micro-plate. Therefore, it is desirable that the fluorescence detection from the micro-plate also include the steps of placing the micro-plate on the base made of transparent material such as glass, irradiating the specimens received in the wells with the stimulating light from the lower side of the micro-plate through the base to excite the fluorescent substance contained in the specimens and to release the fluorescent light from the fluorescent substance, and photoelectrically detecting the released light by the detecting means provided below the base.
However, even if the image carrier is placed on the flat base, it is difficult to make the bottom surface of the image carrier contacting the base completely flat. Thus, gaps filled with air and having uneven thickness (vertical length) are often formed between the bottom surface of the image carrier and the top surface (floor) of the base.
Since the stimulating ray directed onto the image carrier from the base is refracted by the gaps, which usually have uneven thickness, the path of the stimulating ray irregularly shifts and its diameter irregularly varies depending on the thickness of the individual gaps.
Thus, the position actually irradiated by the stimulating ray shifts from the position intended to be irradiated by a distance depending on the thickness of the gap concerned. As pointed out above, the thickness of the gaps is uneven and therefore, the distance between the position actually irradiated and the position intended to be irradiated is not constant in the same image carrier. Further, due to the change in beam diameter, the irradiation power density is not constant in the same image carrier. Accordingly, distortion and irregularity appear in the produced fluorescence image.
The stimulating ray is reflected by both the image carrier and the flat base. The stimulating ray reflected by the image carrier interferes with the stimulating ray reflected by the flat base. Therefore, if the thickness of the gaps is not uniform, the degree of the interference between two reflected lights is different in the same image carrier, resulting in irregular interference in the image carrier.
Such distortion and irregularity are serious problems especially in image reading from a micro-plate. Specifically, the bottom surface of the micro-plate or the surface facing the base when the image carried on the micro-plate is read may be not flat but have multiple recesses or grooves. Therefore, when the fluorescence images of the specimens labeled with the fluorescent substance and received in the wells of the micro-plate are to be read from the base side, since the thickness of the air presenting in the recesses or grooves between the micro-plate and the base is not constant, distortion and irregularity appear in the produced image and the accuracy in image reading is degraded.
It is therefore an object of the present invention to provide an image reading apparatus which can be used in a fluorescence detecting system and can accurately read the image regardless of the kind of image carrier.
The above and other objects of the present invention can be accomplished by an image reading apparatus comprising at least one laser stimulating ray source for emitting a laser beam, a base made of transparent material and bearing thereon an image carrier which carries a fluorescence image formed by a fluorescent substance which can be excited and release fluorescent light in response to irradiation by the laser beam, a laser beam scanning means for upwardly scanning a bottom surface of the image carrier borne on the base with the laser beam emitted from the laser stimulating ray source through the base, light detecting means provided below the base for photoelectrically detecting fluorescent light released from the image carrier and an image carrier receiving portion provided on the base and containing a substance having a refractive index larger than that of air and closer to that of the image carrier than that of air, the substance filling gaps between the image carrier and the base.
In a preferred aspect of the present invention, the substance having a refractive index larger than that of air and closer to that of the image carrier than that of air is a liquid.
In another preferred aspect of the present invention, the substance having a refractive index larger than that of air is water.
In a further aspect of the present invention, the image carrier comprises a micro-plate made of transparent material and having a plurality of wells for receiving specimens labeled with a fluorescent substance.
In a further aspect of the present invention, the image carrier comprises a gel support including a specimen labeled with a fluorescent substance.
In a further aspect of the present invention, the base is provided with retaining means for fixing the image carrier.
In a further aspect of the present invention, the at least one laser stimulating ray source is a laser stimulating ray source for emitting a laser beam having a wavelength between 470 nm and 480 nm.
In a further aspect of the present invention, the at least one laser stimulating ray source is a laser stimulating ray source for emitting a laser beam having a wavelength of 633 nm or 635 nm.
In a further aspect of the present invention, the at least one laser stimulating ray source is a laser stimulating ray source for emitting a laser beam having a wavelength between 530 nm and 540 nm.
As termed with respect to the present invention, the phrase xe2x80x9cthe image carrier carries an image of a fluorescent substancexe2x80x9d includes the case where the image carrier carries an image of a specimen labeled with a labeling substance and the case where the image carrier carries an image of a fluorescent substance obtained by combining an enzyme with a specimen labeled with a labeling substance, causing the enzyme to contact a fluorescent substrate and transforming the fluorescent substrate to a fluorescent substance. In the present invention, the term xe2x80x9cimage carrierxe2x80x9d includes an object such as a gel support which contains a specimen labeled with a fluorescent substance, an object such as a micro-plate holding a specimen labeled with a fluorescent substance in a well or an object such as a piece of gel attached to a glass plate.
In the present invention, the fluorescent dye employed for labeling the specimen to form an image to be carried in an image carrier and read by stimulating it using a laser beam having a wavelength between 470 nm and 480 nm, may be any type of fluorescent dye insofar as it can be stimulated by a laser beam having a wavelength between 470 nm and 480 nm. However, preferably employed fluorescent substances stimulable by a laser beam having a wavelength between 470 nm and 480 nm include Fluorescein (C.I. No. 45350), Fluorescein-X indicated by the structural formula (1) shown below, YOYO-1 indicated by the structural formula (2), TOTO-1 indicated by the structural formula (3), YO-PRO-1 indicated by the structural formula (4), CY-3 (registered trademark) indicated by the structural formula (5), Nile Red indicated by the structural formula (6), BCECF indicated by the structural formula (7), Rhodamine 6G (C.I. No. 45160), Acridine Orange (C.I. No. 46005), SYBR Green (C2H6OS), Quantum Red, R-Phycoerytlmn, Red 613, Red 670, Fluor X, FAM, AttoPhos, Bodipy phosphatidylcholine, SNAFL, Calcium Green, Fura Red, Fluo 3, AllPro, NBD phosphoethanolamine and the like.
In the present invention, the fluorescent substance employed for labeling a specimen to form an image to be carried in an image carrier and read by stimulating it using a laser beam having a wavelength of 633 nm or 635 nm may be any type of fluorescent dye insofar as it can be stimulated by a laser beam having a wavelength of 633 nm or 635 nm. However, preferably employed fluorescent substances stimulable by a laser beam having a wavelength of 633 nm or 635 nm include CY-5 (registered trademark) indicated by the structural formula (8), Allphycocyanin and the like.
Moreover, in the present invention, the fluorescent substance employed for labeling a specimen to form an image to be carried in an image carrier and read by stimulating it using a laser beam having a wavelength between 530 nm and 540 nm may be any type of fluorescent dye insofar as it can be stimulated by a laser beam having a wavelength between 530 nm and 540 nm. However, preferably employed fluorescent substances stimulable by a laser beam having a wavelength between 530 nm and 540 nm include CY-3 (registered trademark) indicated by the structural formula (5), Rhodamine 6G (C.I. No. 45160), Rhodamine B (C.I. No45170), Ethidium Bromide indicated by the structural formula (9), Texas Red indicated by the structural formula (10), Propidium Iodide indicated by the structural formula (11), POPO-3 indicated by the structural formula (12), Red 613, Red 670, Carboxyrhodamine (R6G), R-Phycoeryhthrin, Quantum Red, JOE, HEX, Ethidium homodimer, Lissamine rhodamine B peptide and the like. 